1. Field of the Invention
The present invention relates to an apparatus and method for platelet multi-function analysis, and, more particularly, to an apparatus and method capable of measuring adhesion and aggregation of platelets within a short period of time using a trace of a blood sample, and a stirring microchip provided in the apparatus.
2. Discussion of Related Art
Thrombosis is a disease in which hemostasis or thrombus develops in blood vessels. When these symptoms appear in the coronary arteries of the heart or the brain blood vessels, heart attack or cerebral infarction is caused. Thus, this is called a “silent killer,” and it has emerged as a major cause of death among modern people. This is problematic because such thrombosis or hemorrhagic symptoms are not diagnosed as genetic defects and have not been clearly identified yet.
Also, the more severe problem is that the morbidity of thrombosis tends to increase rapidly due to genetic and acquired factors. Therefore, there has been a demand for apparatuses and methods capable of quantitatively checking up thrombosis or hemorrhagic symptoms and making early diagnosis and prognosis based on the quantitative checkup.
Various factors associated with the hemostasis mechanism are present in damaged blood vessels. In this case, these factors have important biochemical and biophysical mechanisms. Among these, it has been known that platelets play a critical role in the hemostasis mechanism. Platelets does not have adhesion to intact blood vessel walls, but function to stop bleeding at a damaged blood vessel wall regardless of any flow conditions through action of both biochemical and biophysical mechanisms.
When the endothelium of blood vessels is damaged, ingredients such as collagen which constitute a material in the endothelium of blood vessels are exposed to the blood stream, and platelets are attached to these ingredients to be activated. The adhesion mechanism of the platelets has different characteristics depending on environments for the blood stream.
Specifically speaking, when a blood flow velocity is high as in arteries and a shear stress applied to the blood vessel walls is high, the platelets are not easily attached to the inner walls of blood vessels. However, since the platelets are activated under the conditions of a high shear flow field, they are easily attached to the blood vessel walls by means of a von Willebrand factor (vWF) that functions to facilitate adhesion to the blood vessel walls. Of course, it has been known that a glycoproteic receptor complex, GPIb-IX-V, which is present in cell membranes of the platelets, facilitates interaction with the vWF so as to attach the vWF to the cell membranes of the platelets.
Such attached platelets attract the same kinds of platelets to facilitate aggregation, thereby resulting in hemostasis, and then reinforcing a hemostatic action due to the presence of fibrin.
However, such functions of the platelets do not always work well, and may work adversely under certain flow conditions or situations. For example, when a blood vessel wall is locally stenosed due to arteriosclerosis, a part of the blood vessel wall through which platelets pass is exposed to a high shear rate so that the platelets can be activated, and adhesion/aggregation of the platelets takes place at the rear part of the stenosed blood vessel wall, thereby inducing thrombosis in which blood vessels are clogged (Nesbitt et al., A shear gradient-dependent platelet aggregation mechanism drives thrombus formation, Nature Medicine, 15:665-675, 2009).
As described above, the platelets and vWF are activated according to the size of blood flow, that is, the shear stress caused by the blood flow, and a hemostasis mechanism is realized due to an increase in adhesivity. It has been known that the shear stress required to activate the platelets or the vWF as described above is equal to and more than 8 Pa, and a shear rate is equal to and more than 5,000 1/s (Ikeda et al., The role of von Willebrand factor and fibrinogen in platelet aggregation under varying shear stress, J. Clinical Investigation, 87; 1234-1240, 1991).
To perform early diagnosis and prognosis of the hemostasis or thrombus symptoms as described above, various apparatuses have been proposed and developed. However, when the various apparatuses are classified using a measurement sensor, there are an electrical measurement system, an optical measurement system, and a system for measuring a hemostasis time.
Various methods and apparatuses have been developed together to subdivide and examine such functions of the platelets. The analysis of platelet functions is very important in discriminating a hemorrhagic disease caused by congenital or acquired platelet dysfunctions from a hemorrhagic disease in which there is no numerical abnormality in platelets. Also, this analysis of platelet functions tends to be increasingly used to determine an increase in hemorrhagic tendency due to an anti-platelet agent used to treat and prevent cardiovascular diseases or test resistance to drugs.
In general, the analysis of platelet functions is often performed to check congenital platelet dysfunctions or used as a preoperative screening test. Particularly, a bleeding time (BT) test system has been used for important analysis to discriminate a hemorrhagic disease caused by congenital or acquired platelet dysfunctions from a hemorrhagic disease in which there is no numerical abnormality in platelets.
The BT test is a test for measuring a bleeding time that was developed approximately 100 years ago and has been used to screen platelet functions. However, the analysis of platelet functions used so tar is difficult to standardize and clinical applicability is low, and thus an invasive method should be used. Thus, there is a demand for an objective method capable of measuring the platelet functions.
In recent years, a platelet function analyzer (for example, PFA-100) used for measuring the functions of the platelets is characterized in that the platelets aggregate by a vWF which is activated at a high shear rate. To measure the functions of the platelets, the whole blood is allowed to flow at a high shear rate through long capillary vessels. Then, a method of measuring gradual clogging of an orifice coated with ADP or epinephrine along with collagen as the platelet aggregate around the orifice using a pressure or flow rate has been performed.
To analyze such functions of the platelets, a test that should absolutely depend on functions of the vWF and is dependent on hematocrit (Hct) should be performed, but an anti-aspirin or anti-clopidogrel test should not be performed. Also, a two-step test is required to analyze the functions of the platelets, which results in an increase in test costs. Also, at least 360 μl of blood is required to analyze such functions of the platelets, a pressure should be measured to maintain a constant flow of blood at a high shear rate, and a syringe pump should also be continuously driven to correct the pressure when the pressure drops. The flow rate slows down as the orifice is gradually clogged. In this case, since the flow rate exponentially slows down, it is difficult to measure an exact closure time according to platelet aggregation, which makes it difficult to expect the closure time using a mathematical technique.
In particular, the blood sample should be exposed to a high shear rate for at least a predetermined time so as to activate the vWF. To do this, a method of flowing blood through a fairly long capillary vessel at a high velocity may be used in PFA-100. However, this method has problems in that it requires a great quantity of blood, and the vWF around the capillary wall having the maximum shear rate may be easily activated but the vWF disposed at a central region of the capillary wall having the minimum shear rate is not activated. As a result, the reproducibility of the test results may be low.
IMPACT from Diamed uses a cone plate-type rotational Couette flow system to apply a constant shear stress to blood filled therein. Therefore, this is a method of measuring an adhesion level of platelets when a high shear stress is applied to the blood. This has a problem in that, like PFA-100, it is highly dependent on concentrations and functions of vWF and fibrinogen.
Verify-NOW (Accumetrics) uses a principle of measuring turbidity as an aggregation level of platelets using an optical sensor. This is a method in which an agonist is mixed with blood and reacted with microbeads whose surfaces are coated with collagen to induce aggregation of platelets in blood, and the aggregation of platelets is measured as turbidity with time. Thus, this method is often used even though it has the same technical problems as a variety of conventional methods of measuring turbidity.